Methods and compositions for treating viral infections

ABSTRACT

The disclosure demonstrates the inhibition of replication of human cytomegalovirus (HCMV) in cultured human embryo skin muscle cells by two separate subclasses of direct-acting smooth muscle relaxing agents alone or in combination with each other. These two subclasses are characterized mechanistically as calcium influx blockers (or calcium channel blockers) and cyclic nucleotide modulators. More specifically, the class of calcium influx blockers is exemplified by the drugs verapamil (and methoxyverapamil), nifedipine (the prototype drug of 1,4 dihydropyridines), and diltiazem. The class of cyclic nucleotide modulators is exemplified by the drugs isobutylmethlyxanthine, papaverine (and its synthetic analog dioxyline), forskolin, and sodium nitroprusside. In addition, the present disclosure demonstrates that agents from one class, e.g., a calcium influx blocker, act synergistically when used in combination with agents from the other class, e.g., cyclic nucleotide modulators. Moreover, the calcium influx blockers are shown to act synergistically when used in combination with alpha interferon.

The government may own rights in the present invention pursuant to NIHcontract NO1-AI42557.

BACKGROUND OF THE INVENTION

The present application is a continuation-in-part of application Ser.No. 601,471, filed Apr. 18, 1984 now U.S. Pat. No. 4,663,317.

The present invention relates to methods and compositions for treatingviral infections. More specifically, the invention is directed towardsthe treatment of viral infections through the use of smooth musclerelaxing agents and agents that block the entry of calcium ions (Ca⁺⁺)into cells. These agents appear to act by blocking replication of thetarget viruses in infected cells.

Advances in the treatment of viral infections have been very slow incoming. Very few efficacious antiviral agents presently exist. A fewagents have been touted for their potentially specific antiviralactivity are in the research and development stage. The clinicalefficacy of these agents, such as interferon and interferon inducers(e.g., polyanionic pyran copolymers and double stranded RNA) have yet tobe reproducibly demonstrated.

A few pharmaceutical agents have shown promise in the treatment ofisolated viral infections, including the use of amantadine in thetreatment of Influenza A₂ strains. The use of the antimetabolities,Idoxuridine and Cytarabine, in antiviral therapy is hampered by a narrowspectrum of activity and potentially severe side effects. Methisazone isreceiving some support for its use against some pox and vacciniastrains. Its use in pox infections is generally limited to prophylaxis.In general, there are presently no efficacious antiviral agents thatdemonstrate a broad spectrum of activity. It now appears that no singlebroad spectrum agent, or family of agents, may be identified as usefulin antiviral treatment. Therefore, research is being directed towardsidentifying antiviral agents with activity against selected viraldiseases.

A novel approach to the treatment of certain viral diseases, includinghuman cytomegalovirus (HCMV), varicella zoster virus, and herpes simplesvirus, is addressed by the present invention. This approach involves therestriction of viral expression and replication in infected cells bycontrolling and modifying the cellular responses to viral infection.

HCMV causes acute and apparently life-long persistent infections of man(T. H. Weller, N. Eng. J. Med. 285: 203-214, 267-274 (1971)). HCMVinfection has been determined the causative agent in a number of birthdefects, including microencephalopathy, hydroencephelopathy andmicrothalmia. Other defects associated with pre-natal HCMV infectionsinclude severe mental retardation, disordered hepatic function, andhyperbilirubinemia. Although the disease is often asymptomatic inchildren and adults, HCMV infections in these groups have been shown toresult in enlargement of the liver and spleen and derangederythropoesis. The disease may remain dormant for years, thenreactivated by unknown causes. Localized and generalized HCMV infectionshave been shown to develop after immuno-suppressive and anti-neoplastictherapy. HCMV is a member of the herpes family of viruses.

The most widely recognized feature of HCMV-induced cytopathology is theformation of distinct nuclear and cytoplasmic inclusions (CI's) (T.Albrecht, T. Cavello, N. L. Cole, and K. Graves, Lab. Invest., 42: 1-7(1980)). Another HCMV cytopathic effect involves the rounding offibroblastic cells beginning within the first several hours afterinfection. By 12-24 hours post-infection, depending on the intensity ofthe infection, nearly all cells are small and rounded.

The novel approach to restrict virus expression and replicationpresented by the present invention is to control the cellular responseto virus infection. Such approaches may be particularly waranted forhuman cytomegalovirus since this virus is an important cause of diseasefor which effective therapeutic agents have not yet been identified.Additionally, as previously noted, HCMV infections present notablechanges in cytopathology which suggest that cytophatic-directed therapymight prove particularly efficacious in the treatment of that disease.

SUMMARY OF THE INVENTION

A method is provided for the treatment of human viral diseases, inparticular HCMV and HSV, through the use of drugs heretofore unknown topossess antiviral activity. These agents can be classified in a broadsense as direct-acting smooth muscle relaxing agents. Two subclasses ofthe direct-acting smooth muscle relaxing agents, the calcium influxblockers and cyclic nucleotide modulators, have shown antiviralactivity.

In particular, a method for treating viral infections in an infectedhost is presented wherein the method includes administering to the hostan effective amount of a calcium influx blocker in combination with aneffective amount of a cyclic nucleotide modulator. Calcium influxblockers are generally represented by the 1,4 dihydropyridines, theveraponoids and diltiazem. The cyclic nucleotide modulators aregenerally represented by the agents, isobutylmethylxanthine, papaverine,dioxyline, sodium nitroprusside, and forskolin. These agents were chosenas representative agents for their particular chemical structural class.For example, sodium nitroprusside is representative of those smoothmuscle relaxing agents known as the nitrates. Similarly, the verapanoidsare represented by, for example, verapamil and methoxyverapamil.Similarly, the drug nifedipine is representative of the class of 1,4dihydropyridines.

In a further aspect of the present invention, a method for treatingviral infections in an infected host is disclosed which includesadministering to the host an effective amount of alpha interferon incombination with an effective amount of a calcium influx blocker. Thiscombination also demonstrates synergistic antiviral activity over andabove either one of these agents antiviral activity when used alone.

The antiviral methods of the present invention may conveniently beperformed utilizing a pharmaceutical composition which comprises aneffective amount of a calcium influx blocker, or a pharmaceuticallyacceptable salt thereof, in combination with an effective amount of acyclic nucleotide modulator, or a pharmaceutically acceptable saltthereof. Such compositions may further include the addition of aneffective amount of alpha interferon. Interferon-containingcompositions, however, may simply include the alpha interferon incombination with an effective amount of a calcium influx blocker.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the total ⁴⁵ CaCl₂ accumulated by CMV-infected cells( - - - ), CMV infected cells in presence of 30 ug/ml verapamil (Δ - - -Δ), and uninfected (o - - - o) THY (a human fibroblast cell line) cells.Tightly confluent monolayers were infected at a MOI (multiplicity ofinfection) of 3 PFU/cell. After 1 h absorption, the virus was decantedand EMEN (Eagle's modified essential medium) with 5% FCS was added. Atdifferent times, post infection, the cells were labeled with ⁴⁵ CaCl₂ 1uCi/ml for 3 min. The labelling media was then removed and cells werewashed twice with ice-cold isotonic sucrose (8.75% w/v). Then cells wereextracted with 1 ml of 0.1N NaOH added to each dish for 2 h.Radioactivity was measured with a Beckman scintillation counter.

FIG. 2 illustrates the intracellular sequestering of ⁴⁵ Ca⁺⁺ inAD169-infected (3 PFU/cell) (- - - -) and mock-infected (- - o - -) THYcells continuously labelled with ⁴⁵ CaCl₂ (1 uCi/ml: specific activity,27.68 mCi/mg). Each datum point represents the mean of thedeterminations for 3 independent cultures processed separately, ±standard error of the mean.

FIG. 3 demonstrates the intracellular free [Ca⁺⁺ ] response to CMVinfection. Confluent monolayers of THY cells were infected with CMV(strain AD-169) at a MOI of 3-5 PFU/cell for 1 h. At selected intervals,the medium was aspirated and the cells were incubated for 1 hr in EMENcontaining 60 ug/ml of Quin-2 AM at 37° C. The cells were washed withPBS and removed from the flasks with 0.25% trypsin, pelleted, washedtwice, and resuspended in a buffer containing 140 mM NaCl, 5 mM KCL₂, 20mM HEPES, 10 mM glucose, 1 mM MgCl₂. The fluorescence was measured witha spectrophotofluorometer at emission and excitation wavelengths of 339and 492 nM, respectively. Titration of free Ca⁺⁺ activity and computeranalysis of the data were done. Each datum point represents the mean ofthe determinations for three or more independent cultures processedseparately, ± the standard error of the mean.

FIG. 4 illustrates the effect of CMV on the rate of ⁸⁶ Rb uptake. THYcells in confluent monolayers were infected with CMV (strain AD-169) ata multiplicity of 2 PFU/cell. At 24 h PI the medium was aspirated andthe cells were incubated for 0, 3, 6, 9, 15 and 30 minutes in EMENcontaining 1 uCi/ml of ⁸⁶ Rb and/or 120 ug/ml of Ouabian. THY cells inthe absence of (- o -) or presence (- Δ -) of Ouabain; CMV-infected THYcells in the absence (- -) or presence of Ouabain; (- -). Each datumpoint represents the mean of the determination for 3 independentcultures processed separately, ± the standard error of the mean.

FIG. 5 shows the effect of CMV on the ouabain-sensitive Na⁺ /K⁺ ATPase.THY cells in confluent monolayers were infected with CMV (strain AD-169)at a multiplicity of 3.5 PFU/cell. At selected times after infection,the medium was aspirated and the cells were incubated for 30 minutes inEMEN containing 1 uCi/ml of ⁸⁶ Rb and/or 120 ug/ml of ouabain.

FIG. 6 demonstrates the inhibition of CMV replication by Ca⁺⁺ influxblockers. Human embryo skin muscle (SM) cells were infected with CMV(strain AD-169, MOI=5 PFU/cell) and treated at 0 h PI with severalconcentrations of diltiazem ( ), verapamil ( ) or nifedipine (o). Virusyields were measured at 96 h PI by plaque assay.

FIG. 7 demonstrates the inhibition of HSV-1 replication by Ca⁺⁺ influxblockers. SM cells were infected with HSV-1 (strain KOS, MOI=3 to 5PFU/cell). Virus yields were measured at 30-36 h PI by plaque assay.Cells were treated at 0 h PI. ( ), verapamil; (o), nifedipine; ( ),diltiazem. Data plotted are the average of 2 or more experiments.

FIG. 8 demonstrates the inhibition of HSV-1 replication by cyclicnucleotide modulators. SM cells were infected with HSV-1 (strain KOS,MOI=3 to 5 PFU/cell). Virus yields were measured at 30-36 h PI by plaqueassay. Cells were treated at 0 h PI. ( ), papaverine; ( ), sodiumnitroprusside; ( ), forskolin. Data plotted are the average of 2 or moreexperiments.

FIG. 9 demonstrates the inhibition of CMV replication by cyclicnucleotide modulators. Human embryo skin muscle (SM) cells were infectedwith CMV (strain AD-169, MOI=3 to 5 PFU/cell) and treated at 0 h PI withvarious doses of cyclic nucleotide modulators. Virus yields weremeasured at 96 h PI by plaque assay. ( ), papaverine; ( ), sodiumnitroprusside; ( ), forskolin. Data plotted are the average 3 or moreexperiments.

FIG. 10 demonstrates the isopyinic centrifugation of DNA isolated fromCMV-infected cells. Human embryo skin muscle (SM) cells were infectedwith CMV (strain AD-169, MOI=3 PFU/cell) and treated with variousconcentrations of papaverine at 0 h PI. Cells were labelled with ³H-thymidine (10 uCi/ml) from 72 to 96 h PI. (A) No papaverine; (B)papaverine 3 ug/ml; (C) papaverine 10 ug/ml; (D) papaverine 30 ug/ml.

FIG. 11 illustrates the structure of isoquinolines and other chemicalsrelated to papaverine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In its most general and overall scope, the present invention embodiesthe realization that viral infections may be successfully treatedthrough the inhibition of cellular calcium fluxes. The antiviral agentsof the present invention appear to act through calcium flux inhibitionwhich, in turn, serves to block viral replication. This viralreplication can most readily be visualized in the case of HCMVinfections where viral infection is accompanied by a characteristic,readily observable, morphologic cascade. The sequence of the cellularresponses exhibited upon HCMV infection is rounding, "contraction,""relaxation," and enlargement. Rounding of cells begins before 5 hourspost-infection (PI) when cells in intermediate stages of rounding areobserved, and continues through 12 to 24 hours PI. At this time thepopulation of those rounded cells with the smallest size is similar indiameter to that of the nucleus of uninfected cells. By 48 hours PI mostinfected cells have "relaxed," partially flattened, and begun toenlarge. At later times, HCMV-infected cells are observed to be muchenlarged. Inhibition of early cellular responses to HCMV infection isachieved by Ca⁺⁺ influx blockers and other smooth muscle relaxingagents.

The present invention utilizes drugs which inhibit the observedmorphologic cascade which cells undergo during HCMV infection. Theinitial response of the cells to HCMV infection, cell rounding, ismediated by the activation of contractile elements within the cell.Likewise, cellular control of these contractile elements is mediated bychanges in the intracellular concentration of calcium ions. Morespecifically, numerous cellular responses to early viral infection areconsidered with a rise in intracellular free Ca⁺⁺. Thus, thesemorphologic responses could be related to a change in plasma membranepermeability and a concomitant Ca⁺⁺ influx.

Thus, in certain aspects, the present invention embodies the realizationthat viral infections may be treated by correcting for symptomaticchanges in cellular morphology. The successful use of smoothmuscle-relaxing agents in reversing the cell rounding induced by HCMV isdemonstrated by direct microscopic examination of infected cells afterdrug treatment. A concomitant inhibition of viral replication isdemonstrated by fold reductions in viral plaque forming units beingreleased from treated cells.

The antiviral agents of the present invention appear to act by eitherinhibiting the influx of extracellular calcium into infected cells(i.e., the calcium influx blockers) or by inhibiting the flux of calciumintracellularly from intracellular calcium "pools". In CMV infectedcells, calcium flux inhibition of either variety results in the observedreturn to morphologic normally. In treating viral infections which donot effect similar cellular morphologic changes, (for example, as inmeasles, influenza and HTLV III (AIDS) infections) there, of course, isno readily observable morphologic reversion as in the case of CMV.However, the fact that the present agents are active in either casesuggests the possibility that calcium sensitive antiviral targets mayexist in addition to, or in combination with calcium sensitivemorphologic structures.

Smooth muscle relaxing agents except their activity by both indirect(e.g., through modification of smooth muscle nerve transmissions) anddirect (i.e., through direct action on the cells) actions. Theindirect-acting smooth muscle relaxing agents are represented by a widevariety of agents exhibiting great variations in mechanisms of action.These indirect-acting smooth muscle relaxing agents have not been testedfor activity in inhibiting virus replication in infected cells.

Two classes of direct-acting agents, the calcium influx blockers (alsoreferred to as calcium channel blockers) and cyclic nucleotidemodulators, as a group demonstrate very high inhibition of viralreplication in infected cells.

The calcium influx blockers are represented by three general chemicalclasses of agents: (1) the 1,4 dihydropyridines (of which the prototypedrug is nifedipine), (2) a second class, the verapanoids, which includesverapamil and methoxyverapamil, and (3) a third class characterized bythe drug diltiazem.

The smooth muscle relaxing agents that exert their action via modulationof intracellular cyclic nucleotide levels are generally classified as towhether or not they affect the enzyme, phosphodiesterase.Phosphodiesterase is the enzyme responsible for metabolism of cyclicnucleotides. The most effective cyclic nucleotide modulators, in termsof inhibiting viral replication, are represented by the agentspaperverine, and dioxyline, a synthetic compound that is both chemicallyand pharmacologically very similar to papaverine. The second class ofsmooth muscle relaxing cyclic nucleotide modulators exert their activitythrough poorly understood mechanisms other than through inhibition ofphosphodiesterase. This class includes the agents forskolin and sodiumnitroprusside.

Treatment of virally infected cells with a combination of smooth musclerelaxing agents in accordance with the present invention can provide agreatly enhanced antiviral efficacy when compared to treatment withindividual agents. In particular, treatments with a combination of acyclic nucleotide modulator (e.g., papaverine) with a calcium influxblocker (e.g., verapamil or nifedipine) demonstrate surprisingsynergistic effects. Administered alone, verapamil (30 mM) resulted in a10-fold inhibition and papaverine (5 mM) resulted in an 18-fold viralinhibition. However, when administered in combination at the sameconcentrations, these agents provided a 112-fold inhibition of viralyields. For the purposes of the present invention, it was felt that thebest evidence available to demonstrate these agents activity in humanswas by demonstrating their ability to inhibit viral replication andproduction in infected human cells in culture.

Although it is contemplated that the present invention may besuccessfully utilized to treat numerous types of viral infections,including those caused by the RNA and DNA viruses, the invention isherein disclosed in terms of its particular efficacy in inhibiting CMVand herpes virus infections, both HSV-1 and HSV-2. These viralinfections, in addition to presenting a formidable health problem forwhich few efficacious agents are present available, present a usefulmodel system for identifying those agents which exert their antiviralactivity through modulation of calcium levels. Thus, the morphologicchanges associated with CMV infection of cells, and the resultantmorphology which accompanies CMV inhibition, provides a useful, readilyobservable indicator of antiviral activity. However, the efficacy of thepresent agents in treating viral infections which do not exhibit asimilarly striking morphologic cascade, e.g., the measles, influenza andHCMV III viruses, suggests that the morphologic effects observed in thetreatment of CMV-infected cells may be secondary to, or in addition to,the underlying antiviral mechanism.

In a typical protocal, human embryo skin muscle cells are first grown toconfluency in Leighton tubes in 1 ml. of Eagle's media supplemented withEarle's salts, 10% fetal calf serum (FCS), and 0.75% sodium bicarbonate.This requires approximately 2-4 days growth. At the end of this time,the tubes contain about 2×10⁵ cells. Once confluent, the growth media isremoved and 0.3 ml. of the virus stock is placed onto the cells (5pfu/cell) and allowed to adsorb for 1 hr. at 37° C. The virus stock isaspirated off and the cell monolayer washed twice with maintenance media(Eagle's MEM supplemented with Earle's salts, 5% FCS, and 0.15% sodiumbicarbonate). The last wash is replaced with 1 ml. of fresh maintenancemedia containing the indicated drug concentration. The drug is alwaysmade up fresh.

The cells are generally treated with drug for a total of 120 hours. Insome experiments, the drug-containing media is replaced every 24 hrs.,and in others, every 48 hrs. This variation has no effect on theresults.

To test the level of virus replication during drug treatment, thetreated cells are quick-frozen in a Revco -70° C. freezer, then putthrough two freeze/thaw cycles. The cell-containing tubes are thensonicated in a "bath" sonicator for 45 seconds. The cell lysates areassayed for infecticity by a standard plaque assay. (Albrecht, T. andWeller, T. H., Am. J. Clin. Path., 73: 648-651 (1980)). The followingtable (Table 1) depicts the results of initial experiments whichdemonstrated activity of the smooth muscle relaxing agents in inhibitingHCMV replication using the foregoing protocal.

                  TABLE 1                                                         ______________________________________                                        Inhibition of HCMV Replication by                                             Smooth Muscle Relaxing Agents*                                                                      % Inhibition of                                                                           Fold                                        Drug       Dose (ug/ml)                                                                             Virus Yield Inhibition                                  ______________________________________                                        Verapamil  1          44.4        1.8                                                    3          27.8        1.4                                                    10         78.3        4.6                                                    30         83.3        6.0                                         Nifedipine 1          34.7        1.5                                                    3          56.1        2.3                                                    10         93.0        14.3                                                   30         99.9         1000                                       Isobutyl-  30         44.0         2.27                                       methylxanthine                                                                (IBMX)                                                                        Papaverine 1          97.2        35.7                                                   3          99.98        5000                                                  10         99.995      20000                                                  30         99.998      50000                                       Forskolin  1          40.9        1.7                                                    3          54.5        2.2                                                    10         88.2        8.5                                         Sodium     1          45.5        1.8                                         Nitroprusside                                                                            3          45.5        1.8                                                    10         60.0        2.5                                                    30         99.3         143                                        ______________________________________                                         *multiplicity of infection = pfu/cell                                    

Further tests of the smooth muscle relaxing agents against two types ofherpes simplex virus (HSV-1 and HSV-2) demonstrate the usefulness of thepresent invention in inhibiting the replication of herpes virus. Theparticular strain of HSV-1 utilized was KOS and HSV-2 was 198. Theactivity of the agents against HSV was determined in the same manner asdescribed above for CMV. Both of the viral strains were grown in humanembryo skin muscle cells in culture and the infected cells were thentransferred to media containing 30 ug/ml of the indicated agent and thePFU determined at 24 hours PI. These results are compiled in thefollowing table (Table 2).

                  TABLE 2                                                         ______________________________________                                        Inhibition of HSV-1 and HSV-2 by                                              Smooth Muscle Relaxing Agents*                                                              % Inhibition of Virus Yield                                     Drug            HSV-1      HSV-2                                              ______________________________________                                        Verapamil       96.1       95.8                                               Papaverine      86.1       67.7                                               Sodium Nitroprusside                                                                          0          53.8                                               ______________________________________                                         *multiplicity of infection = 5 pfu/cell                                  

The discussion and experimental examples which follow, provide a morein-depth disclosure of the present invention and is meant to providethose of skill in the art with a greater understanding and appreciationof the mechanistic antiviral function of the agents presently disclosed.

A. Inhibition of the cellular response to cytomegalovirus infection

1. Sequence of cellular responses to CMV infection

Following human embryonic fibroblasts infection with CMV and otherviruses, the infected cells demonstrate a progression of cytopathiceffects (Albrecht et al., 1983), beginning with rounding of the cells by5 hours post-infection (PI). In the course of rounding, the cells assumean intermediate form, which raised the possibility that these cellsmight be undergoing a contractile-like response. This concept wassupported by the finding that the rounded cells decreased in diameteruntil at 24 h PI the cells with the smallest size were approximately thesize of the nucleus of uninfected cells. By 48 h PI, the cells "relax,"flatten, and enlarge. During the next several days, the cellsprogressively enlarge and develop the nuclear and cytoplasmic inclusionscharacteristic of viral infection.

2. "Relaxation" and cytomegaly do not require CMV DNA synthesis

In cytomegalovirus infected cells, DNA synthesis begins between 12 and16 h PI. Thus, rounding and "contraction" of CMV-infected cells(beginning before 5 h PI) are, by definition, early events in the virusreplication cycle, since they occur before the onset of virus DNAsynthesis; "relaxation" and enlargement are presumably "late" events,since they occur only after the time of onset of virus DNA synthesis. Todetermine if "relaxation" and enlargment required CMV DNA synthesis, SMcells were infected and then treated with 30 ug/ml of cytosinearabinoside (Ara-C) which blocks virus DNA synthesis. Ara-C failed toblock either "relaxation" or enlargement. These data indicate thatcytomegaly, although a late event, does not require virus DNA synthesisand is the result of early CMV gene expression.

3. The effect of Ca⁺⁺ influx blockers on the development and progressionof cytomegaly and nuclear inclusions

The previous study demonstrated that late cellular responses to CMVinfection such as "relaxation" and cytomegaly, did not require late CMVgene expression. Since early cellular responses such as rounding and"contraction" are inhibited by Ca⁺⁺ influx blockers (e.g., verapamil,nifedipine), it was possible that late responses would also be inhibitedby these drugs. Treatment of CMV-infected cells with either verapamil ornifedipine at 5 h PI inhibited later cellular responses. Inverapamil-treated cells, NIs were present in most nuclei, although theirsize was reduced relative to untreated controls. These data indicatethat the development and progression of CMV NIs are dependent on a CA⁺⁺influx as are other cellular responses to CMV infection.

4. The effect of cyclic nucleotide modulators on the development andprogression of CMV-induced late cellular responses

The effect of cyclic nucleotide modulators on the development andprogression of cytomegaly and NIs was measured by microscopicexamination of CMV-infected, H & E-stained SM (human enbryo skin musclecells) or THY cells. The cyclic nucleotide modulating drugs were addedat 5 h PI which permitted early CMV-induced cellular responses such ascell rounding and "contraction" to occur. As displayed in Table 3,cyclic nucleotide modulating drugs added at 5 h PI inhibited"relaxation," development of cytomegaly, and the formation andprogression of NIs measured at 48 and 72 h PI. These drugs, which arewell recognized for increasing the concentration of cAMP and/or cGMP inother tissues, were potent inhibitors of late cellular responses to CMVinfection.

                  TABLE 3                                                         ______________________________________                                        Inhibition of CMV-Induced Cytomegaly and Nuclear                              Inclusions by Cyclic Nucleotide Modulators                                           Effect                                                                        on Cyclic Inhibition of Cellular Responses.sup.4                              Nucleotides.sup.3                                                                       Relax-             Nuclear                                   Treatment.sup.2                                                                        cAMP    cGMP    ation Cytomegaly                                                                             Inclusion                             ______________________________________                                        Papaverine                                                                             [ ]     [ ]     +++   ++++     ++++                                  IBMX     [ ]     [ ]     ++    +++      ++++                                  Forskolin                                                                              [ ]             ++++  ++++     ++++                                  Nitroprus-       [ ]     --    --       +++                                   side                                                                          ______________________________________                                         .sup.1 MOI = 5 PFU/cell.                                                      .sup.2 A dose of 3 × 10.sup.-5 g/ml of each drug was used. All drug     were added at 5 h PI.                                                         .sup.3 This information is derived from studies in other tissues. [ ],        increased concentration.                                                      .sup.4 Determined at 48 and 72 h PI. +, 1-25% inhibition; ++, 26-50%          inhibition; +++, 51-75% inhibition; ++++, 76-100% inhibition; -, no           inhibition.                                                              

B. Ca⁺⁺ responses to CMV infection

1. The effect of CMV on Ca⁺⁺ fluxes in human fibroblasts

Measurement of ⁴⁵ Ca⁺⁺ in infected and mock-infected cells following a30 min. incubation period in media containing ⁴⁵ CaCl₂ (1 uCi/ml),suggested that intracellular [Ca⁺⁺ ] increases in CMV-invected cells by1 h post-infection (FIG. 1). This Ca⁺⁺ influx declined by 3 h PI.Significant differences in ⁴⁵ Ca⁺⁺ influx were not noted between thevirus-infection and the control cells from 12 to 24 h PI. This influxwas significantly inhibited in the presence of 30 ug/ml of verapamil.Although these results were reproducible, the experimental data weredependent on several variables. As the multiplicity of infection (MOI)was deceased, the time at which the maximum ⁴⁵ Ca⁺⁺ influx occurred wasdelayed and the amount of radioactivity was decreased. For example, at amultiplicity of 1 PFU/cell, the maximum influx was observed at 8 h PI.If the control cells were stimulated at 0 h by replacing the cell lysatewith fresh rather than spent medium, when a ⁴⁵ Ca⁺⁺ influx was alsoobserved in the control cells. Although the influx obtained under thesealtered conditions occurred at the same time in infected and controlcells, the magnitude of the influx in infected cells was about 2-foldgreater.

2. Sequestering of Ca⁺⁺ in CMV-infected cells

The ability of CMV to increase the amount of Ca⁺⁺ sequestered inTCA-precipitable macromolecules was examined over a similar timeinterval. A rapid and substantial increase in ⁴⁵ Ca⁺⁺ radioactivityassociated with the TCA-precipitable fraction of infected cells wasobserved (FIG. 2). These data indicate that CMV is able to increasetotal cellular and bound cellular Ca⁺⁺ early after infection.

3. Effect of CMV on intracellular free (IF) [Ca⁺⁺ ]

Since many cellular mechanisms are regulated by changes in theintracellular levels of "free" calcium (IF [Ca⁺⁺ ]), the IF [Ca⁺⁺ ] ininfected and uninfected cells were measured using the fluorescent Ca⁺⁺indicator, Quin-2. Quin-2 fluorescence increased by about 100% as earlyas 12 h PI, indicating a large increase in IF [Ca⁺⁺ ]. This risecontinued and then leveled off by 24 h PI (FIG. 3) at which time thefluorescence of infected cells was approximately 3 times more than inuninfected cells.

4. Relationship of CMV gene dosage to IF [Ca⁺⁺ ]

To determine the effect of CMV gene dosage on the CMV-induced increasein IF [Ca⁺⁺ ], cells were infected with different multiplicities ofinfection (MOIs) (10, 3.5 and 1 PFU/cell). The increase in fluorescenceunits (FU) in infected cells relative to the control cells by 24 h PIwas 67.5, 45.8 and 20.7 percent, respectively (Table 4). As the FU areproportional to the IF [Ca⁺⁺ ], these results indicate that the increasein the CMV-induced IF [Ca⁺⁺ ] is dependent on the virus gene dosage.

                  TABLE 4                                                         ______________________________________                                        Effect of the CMV Gene Dosage on Intracellular Free [Ca.sup.++ ]                                  Fluorescence                                                                              Percent                                       Sample              units*      change                                        ______________________________________                                        Uninfected cells    38.6                                                      CMV-infected cells (10 PFU/cell)                                                                  64.6        +67.5                                         CMV-infected cells (3.5 PFU/cell)                                                                 56.3        +45.8                                         CMV-infected cells (1 PFU/cell)                                                                   46.6        +20.7                                         ______________________________________                                         *24 hour postinfection.                                                  

5. Effect of metabolic inhibitors and CMV-inactivation on theCMV-induced IF [Ca⁺⁺ ] response

It was additionally determined that active CMV was required for theobserved increase in the IF [Ca⁺⁺ ]. Thus, when CMV was inactivated byheat (56° for 1 h), preincubation with CMV-specific antisera (37° C. for1 h), or exposure to UV light (7.2×10⁴ ergs/mm²), the fluorescence wasless that that obtained with control cells infected with untreated CMVstock. Inhibition of the fluuorescence increase by CMV inactivation was96, 60 and 74 percent, respectively, at 24 h PI (Table 5). Theinhibition of the fluorescence increase by CMV-inactivation indicatesthat the CMV-induced increase in IF [Ca⁺⁺ ] is dependent upon a viableCMV genoma. As shown in Table 5, cycloheximide (10 ug/ml) inhibited theCMV-induced Quin-2 fluorescence increase by 84%. Cordycepin (20 ug/ml),an inhibitor of mRNA polyadenylation, had a similar effect, inhibitingthe increase in IF [Ca⁺⁺ ] by 62%. Thus, protein synthesis and competentmRNA were also required for the induction of the increase in IF [Ca⁺⁺ ].

                                      TABLE 5                                     __________________________________________________________________________    Summary of the Effects of Metabolic Inhibitors and CMV                        Inactivation on Intracellular Free [Ca.sup.++ ] in                            Human Embryonic Thyroid Cells                                                                                  % inhibition                                                       Fluore-                                                                            % change                                                                            of                                                                 scence                                                                             of fluo-                                                                            fluorescence                                 Cytomegalovirus.sup.1                                                                  Treatment    units.sup.2                                                                        rescence                                                                            change                                       __________________________________________________________________________    Experiment 1                                                                  -        None         41                                                      +        None         92   110                                                +        3-deoxyadeno-                                                                              54   3     79                                                    sine.sup.3                                                           -        3-deoxyadeno-                                                                 sine                                                                 Experiment 2                                                                  -        None         42                                                      +        None         79   88                                                 +        Cyclohexi-   48   14    84                                                    mide.sup.4                                                           -        Cyclohexi-        9.5                                                         mide                                                                 Experiment 3                                                                  -        None         26                                                      +        None         49   88                                                 +        Virus stock  27   4     96                                                    heated to                                                                     56° for 1 h before infection                                  Experiment 4                                                                  -        None         42                                                      +        None         72   71                                                 +        Virus stock  54   29    59                                                    reacted with                                                                  CMV-specific serum.sup.5                                             +        Virus stock  73   74    -4                                                    reacted with                                                                  VZV-specific serum.sup.6                                             -        CMV specific 43   2                                                           serum                                                                Experiment 5                                                                  -        None         56                                                      +        None         75   34                                                 +        Inactivation 60   7     79                                                    of virus stock                                                                with UV-light                                                        __________________________________________________________________________     .sup.1 MOI = 3 to 5 PFU/cell                                                  .sup.2 Measured at 24 h PI                                                    .sup.3 20 ug/ml added to cultured THY cells at 0 h PI                         .sup.4 10 ug/ml added to cultured THY cells at 0 h pI                         .sup.5 Human convalescent serum CMV positive, VZVpositive by                  immunofluorescence and complement fixation assays.                            .sup.6 Human convalescent serum CMVnegative, VZV positive by                  immunofluorescence and complement fixation assays.                            .sup.7 7.2 × 10.sup.4 erg/mm.sup.2.                                

6. The phase of CMV expression responsible for induction of increased IF[Ca⁺⁺ ].

Experimental results suggested that the Ca⁺⁺ influx occurred early inthe replication cycle of CMV. The increase in IF [Ca⁺⁺ ] and themorphologic cellular response also appear to begin at early times. Tomore precisely determine the phase of CMV gene expression responsiblefor the IF [Ca⁺⁺ ] response (increase), protein synthesis was blocked ininfected cells with cycloheximide (CH) and then the cells were releasedfrom the translational block in the presence or absence of a"transcription block" (3'-deoxyadenosine). Data from representativeexperiments are summarized in Table 6. At 24 h PI, about 54% morefluorescence was observed in infected than in noninfected cells.Addition of CH from 0-6 h PI reduced the increase in fluorescence toabout 27%. The presence of 3'-deoxyadenosine from 6 to 24 h PI did notappreciably affect the level of fluorescence beyond that expected wheretranscription of a gene begins in the immediate early phase andcontinues into the early phase, suggesting that the increase inintracellular free Ca⁺⁺ was the result of immediate early CMV geneexpression.

                  TABLE 6                                                         ______________________________________                                        Phase of CMV gene expression responsable for induction                        of increased intracellular free [Ca.sup.++ ]                                                                      %                                                                             inhibition                                                  Fluore-  % change of fluore-                                Treatment         scence   of fluore-                                                                             scence                                    Virus   Drug          units.sup.1,2                                                                          scence change                                  ______________________________________                                        None                      41                                                  CMV.sup.3                 63     +53.6                                        CMV   +     Cycloheximide 52     +27/9  42.5                                              (0-6 h)                                                           None        Cycloheximide.sup.4                                                                         36.5   -11                                                      (0-6 h)                                                           CMV   +     Cycloheximide 45.5   +15.9  26.7                                              (0-6 h)                                                                 +     3'-deoxyadenosine.sup.5                                                       (6-24 h)                                                                      Cycloheximide 37.5    -8.5                                                    (0-6 h)                                                                 +     3'deoxyadenosine                                                              (6-24 h)                                                          ______________________________________                                         .sup.1 Measured at 24 h postinfection.                                        .sup.2 Human embryo thyroid fibroblasts.                                      .sup.3 MOI 3 PFU/ml                                                           .sup.4 Cycloheximide 10 ug/ml                                                 .sup.5 3'-deoxyadenosine (cordycepin) 20 ug/ml.                          

To more definitively determine the phase of CMV gene expressionresponsible for the increase in IF [Ca⁺⁺ ] more stringent conditionswere used. Infected cells were treated with CH from -2 h PI through thetime of virus absorption, and up to 2, 4, or 6 h PI. Then the CH waswashed out and the cells were maintained through 24 h PI in the presenceor absence of 3'-deoxyadenosine. Data summarized in Table 7 indicatethat there was no significant difference between the Quin-2 fluorescenceof CMV-infected cells in the presence or absence of 3-deoxyadenosine,indicating that the CMV-induced rise in intracellular free [Ca⁺⁺ ] is animmediate early function.

                  TABLE 7                                                         ______________________________________                                        Phase of CMV Gene Expression Responsible for Induction                        of Increased Intracellular Free [Ca.sup.++ ]                                                                 % change                                       Treatment                      in floure-                                                       3'-deoxy- Fluorescence                                                                           scence                                   CMV.sup.1                                                                           Cycloheximide.sup.2                                                                       adenosine.sup.3                                                                         units    units                                    ______________________________________                                        -     --          --        33.5     --                                       +     --          --        50       49.3                                     +     -2 to 2 hr. --        47       40.3                                     +     -2 to 2 hr. 2 to 24 hr.                                                                             50       49.3                                     +     -2 to 4 hr. --        45       34.3                                     +     -2 to 4 hr. 4 to 24 hr.                                                                             51       52.2                                     -     -2 to 6 hr. --        29       -13.4                                    +     -2 to 6 hr. --        41       22.4                                     -     -2 to 6 hr. 6 to 24 hr.                                                                             35       4.5                                      +     -2 to 6 hr. 6 to 24 hr.                                                                             47       40.3                                     -     -2 to 24 hr.                                                                              --        33       0                                        +     -2 to 24 hr.                                                                              --        27       -19.4                                    -     --          0-24      34       0                                        ______________________________________                                         .sup.1 MOI 3 PFU/ml.                                                          .sup.2 Cycloheximide 10 ug/ml.                                                .sup.3 3'deoxyadenosine (cordycepin) 20 ug/ml.                                .sup.4 Measured at 24 hr. postinfection.                                 

7. Conservation of the capacity to induce an IF [Ca⁺⁺ ] response amongCMV strains

The effect of CMV laboratory strains (AD169, Davis and C-87) on IF [Ca⁺⁺] was compared. The increases in fluorescence level obtained weresimilar among the three laboratory strains. Increases in fluorescencefrom 82% to 150% were observed. these results indicate that the IF [Ca⁺⁺] response is not unique to strain AD-169, and that the ability of CMVto induce an increase in IF [CA⁺⁺ ] is conserved among other CMVstrains.

8. Effect of Ca⁺⁺ influx blockers on CMV-induced IF [Ca⁺⁺ ]

The sensitivity of the CMV-induced rise in IF [Ca⁺⁺ ] to recognized Ca⁺⁺influx blockers (verapamil and nifedipine) was tested. Addition toverapamil (60 ug/ml) or nifedipine (60 ug/ml) at 0 h PI inhibited theCMV-induced increase in fluorescence when measured at 24 or 48 h PI.While verapamil inhibited the percent change in fluorescence by 75% and51% at 24 and 48 h PI, respectively; nifedipine inhibited thefluorescence increase by 89% and 28%, respectively. Diltiazem (80ug/ml), another Ca⁺⁺ influx blocker, inhibited the increase in IF [Ca⁺⁺] by almost 100% by 24 h PI, while nifedipine inhibited the fluorescenceby 112% with the same experimental conditions.

9. Effect of papaverine on CMV-induced IF [Ca⁺⁺ ]

Since CMV-induced cytopathology and CMV replication are Ca⁺⁺ dependent,the effect of papaverine on the CMV-induced IF[Ca⁺⁺ ] response wastested at various times PI. Papaverine inhibited the CMV-inducedenhancement of IF[Ca⁺⁺ ] as measured by Quin-2 fluorescence. The percentinhibition of fluorescence response was 24% by five hours PI and wasmaximal by 48 h PI approaching 100%. These data suggest the possibilitythat the effect of papaverine on CMV replication and CMV-inducedcytopathology is through control of the IF[Ca⁺⁺ ] response.

From the studies of the cytopathologic responses of cells to CMVinfection, it was suggested that papaverine inhibited the development ofNIs and cytomegaly. To determine if papaverine treatment actually causeda decrease in intracellular free [Ca⁺⁺ ] in CMV-infected cells, theQuin-2 fluorescence in CMV-infected cells was measured in the presenceor absence of 30 ug/ml of papaverine. At this papaverine dose, theQuin-2 fluorescence was inhibited at 24 and 48 h PI by 95% and 103%,respectively. The results suggest that some of the effect of papaverineon the cellular response to CMV and on virus replication could be due toan inhibition of the IF [Ca⁺⁺ ] response.

10. Similarities between the effect of the Ca⁺⁺ ionophore A23187 and CMVon human fibroblasts

Since the late cellular responses to CMV infection were found to beinduced by a Ca⁺⁺ influx, then it is possible that other agents such ascalcium ionophores might induce similar responses in SM and THY cells.Therefore, SM and THY cells were treated with the Ca⁺⁺ ionophore A23187(3×10⁻⁵ M). Enlarged cells resulted after 5 min exposure to theionophore. The involvement of a Ca⁺⁺ influx in cellular responses to CMVinfection is thus consistent with this observation and the previousobservations of inhibition of the late cellular responses to CMV by Ca⁺⁺influx blockers and cyclic nucleotide modulators.

C. Na⁺ responses to CMV infection

1. The effect of Na⁺ entry blockers on CMV-induced cytomegaly

It was hypothesized that an increase in intracellular sodium levels [Na⁺] secondary to the CMV-induced Ca⁺⁺ influx would likely be followed byentry of water into the cell leading to cell enlargement. If this werethe case, then Na⁺ entry blockers such as amiloride would be expected toinhibit enlargement. Indeed, it was found that when amiloride (300 uM)was added at 12 h PI, CMV-infected cells failed to enlarge to the sameextent as untreated CMV-infected controls. In addition, nuclearinclusions were considerably decreased in size at 72 h PI.

2. The effect of CMV on the rate of ⁸⁶ Rb uptake via the Na⁺ /K⁺ pump

Similarly, it was hypothesized that the CMV-induced Ca⁺⁺ influx isassociated with or followed by a Na⁺ influx that would bring about theentry of water into the cell leading to cell enlargement. Since the cellvolume is controlled by the ouabain-sensitive Na⁺ /K⁺ pump, the activityof the pump was messured by measuring the ouabain-sensitive ⁸⁶ Rb uptakein CMV-infected cells in the presence and absence of ouabain. FIG. 4shows the rate of ⁸⁶ Rb uptake during a 30 min interval in CMV-infectedcells at 24 h PI. This experiment shows that the rate of uptake overthis period is linear.

3. The effect of CMV on the ouabain-sensitive Na⁺ /K⁺ ATPase

FIG. 5 demonstrates the effect of CMV on the activity of the Na⁺ /K⁺ATPase (pump). There is an initial phase of inhibition in the activityof the pump which is maximal by 12 to 24 h PI, which is then followed bystimulation of the activity of the pump that starts about 48 h PI andcontinues through 96 h PI. The enhancement of the activity of the pumpcoincides with the phase of cell enlargement after CMV-infectionsuggesting that cytomegaly might be due to increased intracellular Na⁺levels.

4. The effect of amiloride on CMV-induced stimulation ofouabain-sensitive ⁸⁶ Rb uptake

Since enhancement of the Na⁺ /K⁺ pump activity is associated withincreased intracellular Na⁺, the effect of amiloride, a Na⁺ entryblocker, was tested on the CMV-induced stimulation phase of the pump.Amiloride (200 uM) was added to CMV-infected cells at 24 h PI and theouabain-sensitive ⁸⁶ Rb uptake was measured at 48 h PI. The apparentstimulation of the CMV-induced ⁸⁶ Rb uptake was inhibited by more than77.6% to level less than that observed in the control uninfected cellsduring this 24 h period in the presence of amiloride. These dataindicate that the CMV-induced stimulation of the pump was Na⁺ dependent.

5. The effect of nifedipine on the ouabain-sensitive Na⁺ /K⁺ pump

To determine if the enhancement of the activity of the Na⁺ /K⁺ pump inCMV-infected cells was Ca⁺⁺ dependent, the effect of nifedipine on theouabain-sensitive ⁸⁶ Rb uptake was measured. Data indicated thatnifedipine (30 ug/ml), when added at 0 h, inhibited the activity of theNa⁺ /K⁺ pump in CMV-infected cells. In the initial phase of CMV-inducedinhibition, the activity of the pump was further depressed from -21% and-18% at 12 h PI to -67% and -83% relative to the cell control,respectively.

During the CMV-induced enhancement phase, the activity of the pump wasinhibited from 37.5% and 37% at 48 and 72 h PI to -39% and -82% relativeto the cell control, respectively. These data suggest that the effect ofCMV on the activity of the Na⁺ /K⁺ pump may be regulated by theintracellular Ca⁺⁺ levels.

6. The effect of Ca⁺⁺ ionophores on the activity of the Na⁺ /K⁺ pump

Since CMV has an initial phase of inhibition of the Na⁺ /K⁺ pump, it wasdetermined whether this effect was possibly Ca⁺⁺ mediated. In thisexperiment, two Ca⁺⁺ ionophores were used to test their effect on theouabain-sensitive ⁸⁶ Rb uptake in uninfected cells. Table 8 shows thatboth Ca⁺⁺ ionophores (A21387, Ionomycin) at a concentration of 1×10⁻⁵ Mwere able to inhibit the ouabain-sensitive counts by 50%.

                  TABLE 8                                                         ______________________________________                                        The effect of Ca.sup.++  Ionophores on Ouabain-Sensitive .sup.86 Rb           Uptake                                                                        Sample               CMP.sup.1                                                                             % change                                         ______________________________________                                        Exp. 1  SM Cells         6412                                                         SM Cells + Ionomycin.sup.2                                                                     3231    -49.6                                        Exp. 2  SM Cells         5998                                                         SM Cells + A23187.sup.2                                                                        2973    -50.4                                        ______________________________________                                         .sup.1 Thirty min. pulselabel from 120 to 150 min. after addition of the      ionophores.                                                                   .sup.2 1 × 10.sup.-5 M                                             

D. Inhibition of CMV and herpes simplex virus replication

1. By Ca⁺⁺ -influx blockers

Ca⁺⁺ influx blockers have been divided into 3 classes based on differingtissue specificities. Verapamil, nifedipine and diltiazem are the mostextensively studied prototypes of the three classes of Ca⁺⁺ influxblockers. FIG. 6 summarizes the results obtained when the effect of arepresentative of each of the three classes of Ca⁺⁺ influx blockers wasstudied on the replication of CMV in SM cells. Nifedipine was the mosteffective of the three Ca⁺⁺ -influx blocking drugs, while diltiazem wasnot effective in inhibiting CMV yields. Inhibition of CMV yields wasgreater than 95% at a concentration with 10⁻⁴ M of either nifedipine orverapamil. Approximately 2×10⁻⁵ M nifedipine or verapamil was needed toobtain 50% inhibition of CMV yield.

HSV-1 replication was somewhat less sensitive to the antiviral effect ofCa⁺⁺ influx blockers than CMV replication (FIG. 7). A dose of 10⁻⁴verapamil inhibited HSV-1 yields by 90%. The same dose of nifedipineresulted in 77.2% inhibition of the HSV-1 yield. Examination of FIG. 7indicates that 50% inhibition of HSV-1 yields was obtained with a doseof approximately 3×10⁻⁵ M (1.7×10⁻⁵ g/ml) nifedipine or 1.6×10⁻⁵ M(7.8×10⁻⁶ g/ml) verapamil. A concentration greater than 10⁻⁴ M (4.5×10⁻⁵g/ml) of diltiazem was required to obtain 50% inhibition of HSV-1yields.

As a first step in increasing our understanding of the mechanism ofantiviral activity of Ca⁺⁺ influx blockers, a kinetic study was done incells infected with HSV-1 and treated with verapamil. Confluent SM cellswere pretreated with 30 ug/ml of verapamil at various times before virusinfection. Virus yields were measured at 33 h PI. Results are shown inTable 9. Pretreatment of cells with verapamil at 24, 12 or 2 h beforevirus infection resulted in no significant inhibition of HSV-1 yields(7-40%). Pretreatment of cells with verapamil and post-treatment aftervirus infection resulted in levels of inhibition of HSV-1 yields similarto post-treatment with verapamil alone. Enhanced inhibition was notobserved following pretreatment. These data suggest that the inhibitoryeffect of verapamil against HSV-1 replication begins only after virusinfection of cells. Establishment of an antiviral state as observed withinterferon treatment seems to be unlikely with verapamil and efficientreplication of HSV-1 apparently requires a C⁺⁺ influx.

                  TABLE 9                                                         ______________________________________                                        Effect of Pretreatment with Verapamil on                                      the Replication of HSV-1                                                                             Precent                                                            Duration of                                                                              inhibition.sup.b                                                                          Fold                                       Type of treatment                                                                         treatment  of HSV yield                                                                              inhibition.sup.c                           ______________________________________                                        Pretreatment alone                                                                        -12 to -1 h                                                                               3          1.03                                                    -3 to -1 h                                                                              39          1.67                                       Pre- and post                                                                             -12 to -1, 89          9.06                                       treatment     0 to 33 h                                                                    -3 to -1, 90          10                                                       0 to 33 h                                                       Posttreatment                                                                               0 to 33 h                                                                              92          12.5                                       ______________________________________                                         .sup.a SM cells were pretreated with 30 ug/ml of verapamil at various         times before virus infection. HSV1 (strain KOS) was applied to the cells      at an MOI of 6.3 PFU/cell. Virus yields were measured at 33 h PI.             .sup.b Calculated relative to untreated control virus yields.                 ##STR1##                                                                     -   2. The effect of increased extracellular [Ca.sup.++ ] on the antiviral     effects of Ca.sup.++ influx blockers

HSV-1 and CMV replication are sensitive to the effect of Ca⁺⁺ influxblockers. In other words, the replication of HSV-1 and CMV may inducethe rapid influx of Ca⁺⁺ into the cell from the extracellular medium.Based on this observation, it was reasoned that the antiviral effect ofCa⁺⁺ influx blockers would be decreased by increasing the concentrationof extracellular Ca⁺⁺. This possibility was tested using Eagle's minimalessential medium (EMEM) with varying concentrations of Ca⁺⁺ as CaCl₂,1.8 mM, 3.6 mM, and 7.2 mM. Four drugs from three classes of Ca⁺⁺ influxblockers were tested in the presence of increased extracellular [Ca⁺⁺ ].Verapamil (30 ug/ml) inhibited HSV-1 yields by 99%. The inhibitoryeffect of verapamil was decreased by increasing extracellular [Ca⁺⁺ ].More than a 2-fold increase in HSV-1 yield was observed with 3.6 mM Ca⁺⁺and almost 4.8-fold more virus was obtained with 7.2 mM Ca⁺⁺. Theresults with nifedipine were similar. 38% and 46% greater virus yieldswith increasing extracellular [Ca⁺⁺ ] of 3.6 and 7.2 mM, respectively.The antiviral effect of gallopamil, a derivative of verapamil, was alsodecreased with 3.6 mM or 7.2 mM Ca⁺⁺. Virus yields were increased by 82%in 3.6 mM or 7.23 mM Ca⁺⁺. Virus yields were increased by 82% in 3.6 mMCa⁺⁺ and by 50% in 7.2 mM Ca⁺⁺. The yields of HSV from cells treatedwith diltiazem were not greatly affected by altering the extracellular[Ca⁺⁺ ] concentration. Less than a 10% increase in virus yield wasobserved in the presence of diltiazem with 3.6 mM or 7.2 mM Ca⁺⁺. Thesedata support the hypothesis that the antiviral effect of Ca⁺⁺ influxblockers is related to their ability to block the influx ofextracellular Ca⁺⁺.

In addition to the recognized Ca⁺⁺ influx blockers, one cyclicnucleotide modulator was tested. Since papaverine has been postulatedalso to affect Ca⁺⁺ fluxes, the effect of increased extracellular [Ca⁺⁺] on papaverine inhibition of HSV yields was examined. Papaverine (30ug/ml inhibited HSV-1 yield by about 85% in the presence of 1.8 mM Ca⁺⁺.By increasing the extracellular calcium concentration, HSV-1 yields wereincreased by 101% (3.6 mM Ca⁺⁺) or 136% (7.2 mM Ca⁺⁺).

3. By cyclic nucleotide modulators

The cyclic nucleotides cAMP and cGMP have been shown to act as secondaryand/or synarchic messengers and to affect Ca⁺⁺ -mediated events. Thus,the effects of several drugs that increase intracellular concentrationsof cAMP or cGMP or both were tested for antiviral activity. FIGS. 8 and9 summarize the results of experiments with HSV-1- and CMV-infectedcells. Papaverine increases the intracellular levels of both cyclicnucleotides by inhibiting the phosphodiesterase activities. Papaverineinhibited HSV-1 replication in a dose-dependent manner (FIG. 8). A doseof 30 ug/ml of papaverine inhibited HSV-1 replication by about 90%. Adose of 10 ug/ml of papaverine resulted in 70% inhibition while a doseof 3 ug/ml of papaverine exerted less than a 50% inhibition of HSVyields. Nitroprusside increases the intracellular level of cGMP, but notof cAMP. Nitroprusside treatment was not as effective as papaverine.Forskolin, which increases the level of cAMP, did not have an inhibitoryeffect on HSV-1 replication. These results, when considered togetherwith those for papaverine and nitroprusside, suggest that the increaseof both cyclic nucleotides may affect the replication of HSV-1, but thatincreases in the level of either cGMP or cAMP alone may not be asimportant as direct effects on Ca⁺⁺ influx or the coordinate increasesof both cyclic nucleotides.

While cyclic nucleotide modulators gave no better inhibition of HSV-1replication than Ca⁺⁺ influx blockers, papaverine showed a dramaticinhibition of CMV replication (FIG. 9). More than a 99.9999% (10⁶ -fold)inhibition of CMV yield was observed with 10⁴ M (3.4×10⁻⁵ g/ml)papaverine. 99% (100-fold) inhibition of CMV yield was obtained at lessthan 10⁻⁵ M of papaverine. Sodium nitroprusside at 10⁻⁴ M (2.6×10⁻⁵g/ml) resulted in about 99% inhibition of CMV yield. Forskolin, as inthe case of HSV-1 infection, was not effective against CMV infection.The drop in virus yield at a concentration of 10⁻⁴ M forskolin might bedue to the toxicity of forskolin at the time of this particularexperiment, but was not a consistent finding. A comparison of data fromFIGS. 8 and 9 suggest that CMV replication is more sensitive than HSV-1replication to the increase of both cyclic nucleotides or cGMP alone.

4. Comparison of the anti-CMV effect of two papaverine preparations

To ensure that the effect of papaverine (Eli Lilly, Indianapolis, IN),was due to papaverine and not due to other elements in papaverinepreparations as supplied by Lilly for injection, the anti-CMV effect oftwo papaverine preparations (papaverine in solution from Eli Lilly,papaverine in solid form from Sigma) was compared. Both preparations ofpapaverine were found to be similarly effective against CMV replication.Apparently the antiviral effect of Lilly's papaverine preparation is dueto papaverine.

5. The antiviral effect of combination treatment with papaverine andCa⁺⁺ influx blockers

Combination antimicrobial therapy has proved to be effective in curingseveral bacterial diseases. Moreover, combination treatment permits thereduction of the dosage of antibiotics to lessen toxicity and to preventthe appearance of antibiotic-resistant strains. With these advantages inmind, combination treatment with papaverine and Ca⁺⁺ influx blockers wastried. Suboptimal doses of each drug were used. The results (Table 10)suggest the possibility of synergistic effects with combinationtreatment of a cyclic nucleotide modulator (e.g.,--papaverine) and acalcium influx blocker (e.g.--verapamil or nifedipine). Verapamil (30uM) resulted in a 10-fold inhibition and papaverine (5 uM) resulted in18-fold inhibition of CMV yields. When both verapamil (30 uM) andpapaverine (5 uM) were used in combination, 112-fold inhibition of CMVyields was obtained. A somewhat greater synergistic effect was observedwith the combination of 30 uM nifedipine and 5 uM papaverine. Diltiazem,however, did not demonstrate synergistic effects when used incombination with papaverine. In fact, less inhibitory effect was evidentwith combination treatment with diltiazem and papaverine than papaverinealone. These results, in part, correlate with the observation thatverapamil and nifedipine were more effective in inhibiting CMVreplication than diltiazem.

                  TABLE 10                                                        ______________________________________                                        Combination Treatment with Papaverine                                         and Ca.sup.++  Influx Blockers                                                       No papaverine With papaverine                                          Ca.sup.++  influx  Fold              Fold                                     blocker  CMV yield Inhibition                                                                              CMV yield                                                                             Inhibition                               ______________________________________                                        None     3.5 × 10.sup.7                                                                              2.0 × 10.sup.6                                                                  18                                       Verapamil                                                                              3.5 × 10.sup.6                                                                    10        3.1 × 10.sup.5                                                                  112                                      Nifedipine                                                                             7.5 × 10.sup.6                                                                    4.8       2.2 × 10.sup.5                                                                  159                                      Diltiazem                                                                              1.2 × 10.sup.7                                                                    3.0       9.7 × 10.sup.6                                                                  3.6                                      ______________________________________                                         Human embryonic skin muscle (SM) cells were infected with CMV (strain         AD169, MOI = 3.0 PFU/cell). After 1 h adsorption at 37° C., the        Ca.sup.++  influx blockers were added to infected cultures to 30 uM in th     presence or absence of 5 uM papaverine. Media were changed at 48 h with       fresh drugs. Virus yields were determined at 96 h PI.                    

7. The relationship between the time of removal of papaverine andinhibition of CMV yields

As shown in Table 11, the antiviral action of papaverine is readilyreversible through at least 6 h PI. The drug is apparently effectivelywashed out since yields from cells washed at 4 and 6 h were enhancedover untreated controls. Even when the drug was washed out at latertimes through 72 h PI, yields from 37% to 14% of the control wereobtained. Thus, the inhibitory effect of papaverine is reversible. Thediminishing yields obtained following reversal of the papaverine blocksuggest that at later times the inhibitory action of papaverine may notbe reversible even though the drug has been effectively removed. Thispossibility will be tested in future studies. Furthermore, it is notclear from this experiment what is the fate of the papaverine-treated,CMV-infected cell. This question will also be answered in futurestudies.

                  TABLE 11                                                        ______________________________________                                        Effect of the Time of Removal of Papaverine on its                            Inhibitory Effect of CMV Replication                                          Time of                                                                       removal  CMV Yields (PFU/ml) Percent of                                       (h PI)   No papaverine Papaverine                                                                              control.sup.b                                ______________________________________                                        None     2.3 × 10.sup.6                                                                        7.8 × 10.sup.2                                                                    0.034                                         2       3.3 × 10.sup.6                                                                        2.1 × 10.sup.6                                                                    64                                            4       3.1 × 10.sup.6                                                                        5.3 × 10.sup.6                                                                    171                                           6       2.7 × 10.sup.6                                                                        4.5 × 10.sup.6                                                                    167                                          12       1.0 × 10.sup.7                                                                        3.7 × 10.sup.6                                                                    37                                           24       2.2 × 10.sup.7                                                                        5.8 × 10.sup.6                                                                    26                                           48       1.5 × 10.sup.7                                                                        3.9 × 10.sup.6                                                                    26                                           72       1.7 × 10.sup.7                                                                        2.4 × 10.sup.6                                                                    14                                           ______________________________________                                         .sup.a SM cells were infected with CMV (strain AD169) at a MOI of 3.6         PFU/cell. Half of the infected cultures were treated with EMEM containing     10 ug/ml papaverine at 0 h PI. At various times after infection, cells        were washed three times and fed with fresh EMEM. CMV yields were              determined at 96 h PI, except for the 72 h PI wash which was harvested at     120 h PI.                                                                     .sup.b Relative to untreated cultures that were washed at same time as        papavinetreated cultures.                                                

E. Stage of CMV replication sensitive to the antiviral effects ofpapaverine

1. Time of addition studies

As papaverine was highly effective in inhibiting CMV replication, thetime point at which the replication of CMV is sensitive to treatmentwith papaverine was determined. Cells were infected with CMV and treatedwith 10 ug/ml of papaverine at various times before or after virusinfection. Pretreatment with papaverine at 24, 12 or 2 h before virusinfection resulted in no inhibition of the CMV yields, nor theenhancement of antiviral activity when followed by post-treatment at 0 hPI. Addition of papaverine at early times after virus infection through6 h PI gave virtually identical inhibitory effects on CMV replication.With the addition of drug after 6 h, the effect of papaverine droppedapproximately 5- to 10-fold with each successive 12 to 24 h delay;however, the addition of papaverine as late as 48 h PI resulted in asignificant inhibition of CMV yields.

2. Viral DNA synthesis

a. HSV-1 DNA synthesis

The time of addition studies suggested this antiviral effect was mostpronounced when papaverine was added before the time of initiation ofCMV DNA synthesis. The possibility that smooth muscle-relaxing agentswere inhibiting viral DNA synthesis was first examined by testing theeffect of Ca⁺⁺ influx blockers or papaverine on the synthesis of DNA incells infected with HSV-1. The experiments were designed so that totalradioactivity from infected cells was representative of the rate ofHSV-1 DNA synthesis because at the time of radioactive labellingcellular DNA synthesis is almost completely shut down. A representativedrug from each of the three classes of Ca⁺⁺ influx blockers was testedfor its ability to inhibit DNA synthesis in SM cells infected with HSV-1strain KOS. Treatment of infected cells with verapamil or nifedipine at30 ug/ml inhibited the incorporation of radioactive thymidine by 51-60%.Diltiazem was not inhibitory. Ca⁺⁺ influx blockers, however, inhibitedthe incorporation of ³ H-thymidine in mock-infected cells moreefficiently than in HSV-1 infected cells. The results with papaverinewere similar to those observed with Ca⁺⁺ influx blockers except thatboth mock-infected cells had similar levels of inhibition of DNAsynthesis.

b. CMV DNA synthesis

In CMV-infected cells, cellular DNA synthesis is stimulated in the earlyphase of virus infection, but during the late phase nearly all DNAsynthesized is of virus origin. Thus, measurement of ³ H-thymidineincorporated during the late phase of CMV infection representsCMV-specific DNA synthesis. Treatment of CMV-infected cells with Ca⁺⁺influx blockers resulted in a substantial decrease in the rate of CMVDNA synthesis. Again, more efficient inhibition of DNA synthesisoccurred in mock-infected cells than in virus-infected cells. The levelof inhibition of DNA synthesis of CMV-infected cells was lower than thelevel of inhibition of CMV yield.

Papaverine is the most potent of the smooth muscle-relaxing agents whichinhibit the replication of CMV. More than 100,000-fold inhibition of CMVreplication usually is observed with 30 ug/ml of papaverine. When theeffect of papaverine on the synthesis of DNA in cells infected with CMVwas tested, however, only a 91% or 11-fold inhibition of DNA synthesiswas observed. Apparently this reduction in the rate of incorporation of³ H-thymidine represented inhibition of DNA synthesis rather than adelay. When a time-course study done with 10 ug/ml of papaverine, nodisplacement of the pattern of CMV DNA synthesis was observed; incontrast, the extent of inhibition increased with elasped time afterinfection and papaverine treatment as illustrated by Table 12.

                  TABLE 12                                                        ______________________________________                                        Effect of Papaverine on the synthesis of DNA in SM                            Cells Infected with CMV: A Kinetic Study                                      Labeling period                                                                          CPM                 Percent                                        (h PI)     No papaverine                                                                              Papervine  inhibition                                 ______________________________________                                         0-24       9315        6302       32                                         24-48      26864        8142       70                                         48-72      108636       22216      80                                         72-96      87508        7786       91                                          96-120    57116        2876       95                                         ______________________________________                                         Human embryo skin muscle (SM) cells were grown on 35 mm dishes and            infected with CMV (strain Ad169) at a MOI of 3.4 PFU/ml. Papervine was        added at 0 h PI to make an initial concentration of 10 ug/ml. Medium was      changed at 48 h PI with fresh papaverine. Concentrated .sup.3 H--thymidin     (specific activity = 20 Ci/mmol) was added at the times indicated after       CMV infection to a final concentration of 10 uCi/ml.                     

The disparity between the levels of inhibition achieved by papverine ofvirus yield and virus DNA synthesis suggested that inhibition of CMVresulted from mechanisms which affected replication steps other thanjust DNA synthesis, or that the papaverine was not being applied at theoptimal time. A time of addition study was designed and done at a MOI of3.4 PFU/cell. Pretreatment of cells with papaverine had no effect on CMVDNA synthesis. Treatment of cells with papaverine at any time from 0 to24 h PI resulted in a similar level of inhibition of DNA synthesis:about 90% to 95%.

In spite of numerous experiments which were performed on CMV DNAsynthesis, it was possible that cellular DNA synthesis was making a muchlarger contribution to the overall rate of ³ H-thymidine incorporationthan predicted. To determine if the radioactivity measured inCMV-infected cells was truly representative of CMV DNA synthesis, viraland cellular DNA were separated using CsCl density gradientcentrifugation. FIG. 10 shows the radioactivity measured in eachfraction collected from the bottom of representative gradients. At 72-96h PI, almost no cellular DNA was found; most of the radioactivity wasfound in a peak with the recognized density (1.716 g/cm³) of CMV DNA.Since from 72 to 96 h PI, CMV DNA synthesis reaches the maximum rate atthis relatively high MOI, it is unlikely that cellular DNA synthesiscontributed sufficiently to the overall rate of ³ H-thymidineincorporation to result in the erroneous interpretation of the earlierdata.

F. Structure-activity relationships of papaverine-related compounds

Since papaverine has proven to be the most potent anti-CMV drug testedin our protocols thus far, studies of the structural specificity of theantiviral action of papaverine were initiated by comparing the antiviralactivities of several compounds which are structurally related topapaverine and commercially available. Glaucine, laudanosine,salsolidine and tetrahydropapaverine were tested. Their structures, aswell as the structure of papaverine, are shown in FIG. 11. All thesedrugs were dissolved in 70% ethanol to make stock solutions with aninitial concentration of 40 mg/ml. Subsequent dilutions were mode withwater to make working solutions of 10 mg/ml.

Results of virus yield assays in the presence or absence of drugs fromthis gourp of isoquinolines are shown in Table 13. No significantinhibition of CMV replication was observed with these drugs. Onlytetrahydropapaverine at the highest concentration resulted in more than10-fold inhibition of CMV replications. Controls consisting of ethanolat a concentration corresponding to that contained in the highestconcentration of drugs tested were examined for antiviral effects and 3-to 4-fold inhibition of CMV replication was observed.

Therefore, tentatively, recognizing that this is a single experiment, itappears that the nitrogen on the isoquinoline ring with an electronavailble for covalent bond formation is critical to the antiviralactivity of papaverine. When hydrogen occupies this site(tetrahydropapaverine), then the inhibitory activity is reduced from99,99982% inhibition to 89% inhibition at equimolar doses of 100 uM.When a methyl group occupies this site (laudanosine), the antiviralactivity is lost almost entirely. The results with salsolidine andglaucine add further support to this view.

                  TABLE 13                                                        ______________________________________                                        Effect of Papaverine-Related Compounds                                        on the Replication of CMV                                                                Dose      CMV Yields Percent of                                    Drugs.sup.a                                                                              (uM)      (PFU/ml).sup.b                                                                           Control                                       ______________________________________                                        None                 1.8 × 10.sup.7                                                                     100                                           Laudanosine                                                                               3        2.0 × 10.sup.7                                                                     111                                                      10        1.0 × 10.sup.7                                                                     56                                                       30        2.4 × 10.sup.7                                                                     133                                                      100       1.3 × 10.sup.7                                                                     72                                            Glaucine    3        2.1 × 10.sup.7                                                                     117                                                      10        1.1 × 10.sup.7                                                                     61                                                       30        2.1 × 10.sup.7                                                                     117                                                      100       1.6 × 10.sup.7                                                                     89                                            Salsolidipine                                                                             3        1.3 × 10.sup.7                                                                     72                                                       10        1.4 × 10.sup.7                                                                     78                                                       30        1.4 × 10.sup.7                                                                     78                                                       100       4.4 × 10.sup.6                                                                     24                                            Tetrahydro-                                                                               3        1.5 × 10.sup.7                                                                     83                                            papaverine 10        1.7 × 10.sup.7                                                                     94                                                       30        4.8 × 10.sup.6                                                                     27                                                       100       1.9 × 10.sup.6                                                                     11                                            ______________________________________                                         .sup.a Drugs were dissolved in 70% ethanol to make an initial                 concentration of 40 mM and subsequently diluted with water to 10 mM.          Further dilutions were made with EMEM to make 100, 30, 10 and 3 uM.           .sup.b SM cells and CMV strain AD169 at a MOI of 3.7 PFU/cell were used.      Medium was changed at 48 h PI and virus yields were determined at 96 h PI                                                                              

G. Inhibition of CMV and HSV-1 Replication by Combined Treatment withInterferon and Ca⁺⁺ Influx blockers

In addition to demonstrating synergistic action when administered toinfected cells in combination with each other, the pharmacologic agentsdisclosed by the present invention exhibit a synergistic antiviral whenadministered in combination with alpha interferon (IFN alpha). This wasdemonstrated by treating infected cells with a combination whichincluded IFN alpha and verapamil. THY cells were treated with 1000 U/mlof recombinant type IFN alpha (obtained from Schering, Kenilworth, N.J.)24 h before CMV infection. After CMV infection the cells were treatedwith 1000 U/ml of IFN alpha and/or 15 ug/ml of verapamil. Media werechanged at 48 h PI. Virus yields were measured at 96 h PI. As seen inTable 14, CMV replication was inhibited by 1.9-fold with 15 ug/ml ofverapamil and by 3.6-fold with 1000 U/ml of IFN alpha. Combinedtreatment with verapamil and IFN alpha gave a 26.3-fold inhibition ofCMV yield. This number is almost 5-fold higher than 5.5 which is the sumof the levels of inhibition of the two drugs alone (3.6 and 1.9).

                  TABLE 14                                                        ______________________________________                                        Inhibition of CMV replication                                                 by IFN alpha and verapamil                                                                  Virus yield.sup.a                                                                        Percent   Fold                                       Treatment     (pfu/ml)   Inhibition                                                                              Inhibition                                 ______________________________________                                        None          4.78 × 10.sup.6                                           IFN a.sup.b   1.34 × 10.sup.6                                                                    72.0      3.6                                        Verapamil.sup.c                                                                             2.50 × 10.sup.6                                                                    47.7      1.9                                        IFN a + verapamil.sup.d                                                                     1.83 × 10.sup.5                                                                    96.2      26.3                                       ______________________________________                                         .sup.a THY cells were infected with CMV (strain AD169, MOI = 4.5              pfu/cell). Media were changed at 48 PI. Virus yields were measured at 96      PI.                                                                           .sup.b 1,000 U/ml of recombinant IFN alpha was used.                          .sup.c 15 ug/ml.                                                         

This apparent synergistic effect was also observed when cells werepretreated with IFN alpha, infected with HSV-1, and post-treated withthe combination of IFN alpha and verapamil (Table 15). 2000 U/ml of IFNalpha alone gave 33-fold inhibition of HSV-1 yields and 30 ug/ml ofverapamil resulted in 33-fold inhibition. Combined treatment of 2000U/ml of IFN alpha and 30 ug|ml of verapamil gave a 500-fold inhibitionof HSV-1 yield.

                  TABLE 15                                                        ______________________________________                                        Inhibition of HSV-1 Replication of                                            IFN alpha and Verapamil                                                                     Virus yield.sup.a                                                                        Percent   Fold                                       Treatment     (pfu/ml)   Inhibition                                                                              Inhibition                                 ______________________________________                                        None          1.2 × 10.sup.7                                            IFN a.sup.b   3.5 × 10.sup.5                                                                     97        33                                         Verapamil.sup.c                                                                             3.5 × 10.sup.5                                                                     97        33                                         IFN a + verapamil.sup.d                                                                     2.5 × 10.sup.4                                                                     99.8      500                                        ______________________________________                                         .sup.a SM cells were infected with HSV1 (strain KOS, MOI = 5 pfu/cell).       Virus yields were measured at 24 h PI.                                        .sup.b 2,000 U/ml of recombinant IFN alpha was used.                          .sup.c 30 ug/ml.                                                              .sup.d 2,000 U/ml of IFN alpha + 30 ug/ml of verapamil.                  

A more detailed study with IFN alpha and verapamil in HSV-1 infectedhuman cells (Table 16) suggests that the synergistic effect may beoptimized when relatively high (more than 333 U/ml) doses of IFN alphawas used in combination with verapamil doses as low as 3.7 ug/ml. A highconcentration of verapamil (33 ug/ml) and lower dose of IFN alpha (111U/ml) resulted in little synergistic effect.

                  TABLE 16                                                        ______________________________________                                        Inhibition of HSV-1 Replication by Combined                                   Treatment of IFN alpha and Verapamil                                          IFN.sup.a                                                                              Verapamil (ug/ml)                                                    (U/ml).sup.b                                                                           0         3.7       11      33                                       ______________________________________                                         0       1.03 × 10.sup.8                                                                   6.25 × 10.sup.7                                                                   8.25 × 10.sup.7                                                                 1.45 × 10.sup.7                             (1)       (1.6)      (1.25)  (7.1)                                   111      3.25 × 10.sup.7                                                                   3.25 × 10.sup.7                                                                   1.83 × 10.sup.7                                                                 1.05 × 10.sup.7                             (3.2)     (3.2)     (5.6)    (9.8)                                   333      2.63 × 10.sup.7                                                                   1.10 × 10.sup.7                                                                   1.08 × 10.sup.7                                                                 1.65 × 10.sup.6                             (3.9)     (9.4)     (9.5)   (62.4)                                   1,000    8.75 × 10.sup.6                                                                   4.25 × 10.sup.6                                                                   4.25 × 10.sup.6                                                                 1.68 × 10.sup.6                             (11.8)    (24.2)    (24.2)  (61.3)                                   ______________________________________                                         .sup.a SM cells were infected with HSV1 (strain KOS, MOI = 5.0 pfu/cell).     Virus yields were measured at 32 h PI.                                        .sup.b IFN's were pre and posttreated.                                   

A combination treatment study was also done with IFN alpha andnifedipine. SM cells were pretreated with 2000 U/ml of IFN alpha for 18h before virus infection. 2000 U/ml of IFN alpha with or without variousdoses of nifedipine was added after virus infection. Virus yields weremeasured at 24 h PI. Table 17 shows synergistic effects when IFN alphawas used in combination with 10 or 30 ug/ml of nifedipine.

                  TABLE 17                                                        ______________________________________                                        Inhibition of HSV Replication by Combined                                     Treatment of IFN alpha and Nifedipine                                                No IFN alpha  With IFN alpha                                           Nifedipine.sup.b                                                                       Virus     Fold      Virus   Fold                                     (ug/ml)  Yield     Inhibition                                                                              Yield.sup.a                                                                           Inhibition                               ______________________________________                                         0       1.36 × 10.sup.8                                                                              6.0 × 10.sup.6                                                                 22.7                                      3       8.75 × 10.sup.7                                                                   1.56      8.03 × 10.sup.6                                                                 16.47                                    10       6.58 × 10.sup.7                                                                   2.07      2.45 × 10.sup.6                                                                 55.6                                     30       4.30 × 10.sup.7                                                                   3.16      9.13 × 10.sup.5                                                                 149                                      ______________________________________                                         .sup.a SM cells were pretreated with 2000 U/ml of IFN alpha for 18 h          before virus infection, and then infected with HSV1 (strain KOS) at a MOI     of 4.8 pfu/cells. IFN alpha was posttreated with or without combination       with nifedipine. Virus yields were measured at 24 h PI.                       .sup.b Nifedipine was dissolved with PEG 400 to make an initial               concentration of 5 mg/ml.                                                

The findings that direct-acting smooth muscle relaxing agents are activein treating virus-infected human tissue culture cells suggest that suchagents will prove useful in treating viral infections in man. The agentsdo not undergo appreciable entero-hepatic metabolism prior todistribution throughout the body, nor do they require metabolism for"activation." Likewise, these viral diseases present similar morphologicchanges in infected cells both in vivo and in vitro. For many years, ithas been shown that in vitro antiviral activity typically correlateswith in vivo activity. In contrast, the main problem has often been thefinding of untoward reactions (toxicities) in vivo that were not seen invitro. Since the present agents are in clinical use, this will not be aproblem. Therefore, it is expected that these agents can be administeredto an infected patient by all routes presently indicated for their use.It is further expected that topical preparations will be active intreating lesions associated with viral infection of this sort.

The instant invention has been disclosed in connection with standardlaboratory procedures used by the applicant. However, it will beapparent to those skilled in the art that variations may be undertakenwithout departing from the spirit and scope of the invention. Morespecifically, it will be apparent that certain agents that are bothchemically and pharmacologically related may be substituted to achievethe observed antiviral effect. For example, methoxyverapamil andverapamil are virtually indistinguishable pharmacologically as arepapaverine and dioxyline and would be expected to give similar results.Additionally, although the present invention is disclosed in terms ofactivity against CMV and the herpes viruses, it is contemplated that thepresent agents are effective in treating other viral infections. Theseand similar substitutes will be apparent to those skilled in the art andare within the spirit and scope of the invention.

What is claimed is:
 1. A method for treating viral infections in aninfected host comprising administering to the host an effective amountof a calcium influx blocker in combination with an effective amount of acyclic nucleotide modulator.
 2. The method of claim 1 wherein thecalcium influx blocker is selected from the group consisting of:(a) a1,4 dihydropyridine; (b) a verapanoid; and (c) diltiazem; andwherein thecyclic nucleotide modulator is selected from the group consisting of:(a) isobutylmethylxanthine; (b) papaverine; (c) dioxyline; (d) sodiumnitroprusside; and (e) forskolin.
 3. The method of claim 2 wherein the1,4 dihydropyridine is nifedipine.
 4. The method of claim 2 wherein theverapanoid is verapamil or methoxyverapamil.
 5. The method of claim 1wherein the calcium influx blocker is selected from the group consistingof:(a) a 1,4 dihydropyridine; (b) a verapanoid; and (c) diltiazem. 6.The method of claim 5 wherein the verapanoid is verapamil ormethoxyverapamil.
 7. The method of claim 5 wherein the 1,4dihydropyridine is nifedipine.
 8. The method of claim 1 wherein thecyclic nucleotide modulation is selected the group consisting of:(a)isobutylmethylxanthine; (b) papaverine; (c) dioxyline; (d) sodiumnitroprusside; and (e) forskolin.
 9. A method for treating viralinfections in an infected host comprising administering to the host aneffective amount of papaverine in combination with an effective amountof verapamil or nifedipine.
 10. The method of claim 1 or 9 furthercomprising administering to host an effective amount of alphainterferon.
 11. A method for treating viral infections in an infectedhost comprising administering to the host an effective amount of alphainterferon in combination with an effective amount of a calcium influxblocker.
 12. The method of claim 11 wherein the calcium influx blockeris selected from the group consisting of:(a) a 1,4 dihydropyridine; (b)a verapanoid; and (c) ditiazem.
 13. The method of claim 12 wherein theverapanoid is verapamil or methoxyverapamil.
 14. The method of claim 12wherein the 1,4 dihydropyridine is nifedipine.
 15. A method for treatingviral infections in an infected host comprising administering to thehost an effective amount of alpha interferon in combination with aneffective amount of verapamil or methoxyverapamil.
 16. A method fortreating viral infections in an infected host comprising administeringto the host an effective amount of alpha interferon in combination withan effective amount of nifedipine.
 17. A pharmaceutical compositioncomprising an effective amount of a calcium influx blocker, orpharmaceutically acceptable salts thereof, in combination with aneffective amount of a cyclic nucleotide moduclator, or pharmaceuticallyacceptable salts thereof.
 18. The composition of claim 17 wherein thecalcium influx blocker is selected from the group consisting of:(a) a1,4 dihydropyridine; (b) a verapanoid; and (c) diltiazem; andwherein thecyclic nucleotide modulator is selected from the group consisting of:(a) isobutylmethylxanthine; (b) papaverine; (c) dioxyline; (d) sodiumnitroprusside; and (e) forskolin.
 19. The composition of claim 18wherein the verapanoid is verapamil or methoxyverapemil.
 20. The methodof claim 18 wherein the 1,4 dihydropyridine is nifedipine.
 21. Thecomposition of claim 17 wherein the calcium influx blocker is selectedfrom the group consisting of:(a) a 1,4 dihydropyridine; (b) averapanoid; and (c) diltiazem.
 22. The composition of claim 21 whereinthe verapanoid is verapamil or methoxyverapamil.
 23. The composition ofclaim 21 wherein the 1,4 dihydropyridine is nifedipine.
 24. Thecomposition of claim 17 wherein the cyclic nucleotide modulation isselected from the group consisting of:(a) isobutylmethylxanthine; (b)papaverine; (c) dioxyline; (d) sodium nitroprusside; and (e) forskolin.25. A pharmaceutical composition comprising an effective amount ofpapaverine, or a pharmaceutically acceptable salt thereof, incombination with an effective amount of verapamil or nifedipine, orpharmaceutically acceptable salts thereof.
 26. A pharmaceuticalcomposition comprising an effective amount of alpha interferon incombination with an effective amount of a calcium influx blocker. 27.The composition of claim 26 wherein the calcium influx blocker isselected from the group consisting of:(a) a 1,4 dihydropyridine; (b) averapanoid; and (c) ditiazem.
 28. The composition of claim 27 whereinthe verapanoid is verapamil or methoxyverapamil.
 29. The composition ofclaim 27 wherein the 1,4 dihydropyridine is nifedipine.
 30. Apharmaceutical composition comprising an effective amount of alphainterferon in combination with an effective amount of verapamil ormethoxyverapamil.
 31. A pharmaceutical composition comprising aneffective amount of alpha interferon in combination with an effectiveamount of nifedipine.